Spatial Proteomics/Transcriptomics

Background:

Idiopathic Multicentric Castleman disease (iMCD) involves systemic inflammation, multicentric lymphadenopathy, and life-threatening multiple organ dysfunction resulting from a cytokine storm often including the pro-inflammatory cytokine interleukin-6 (IL-6). Though the etiology of iMCD is unknown, an IL-6–mediated cytokine storm is thought to drive disease pathogenesis in some iMCD patients; however, only 34% of patients responded to anti-IL-6 therapy with siltuximab in the phase II trial. With limited treatment options, 60% of iMCD patients die within 10 years of diagnosis, secondary to systemic inflammation and organ dysfunction. More recently, the Fajgenbaum lab and others have further classified iMCD into 2 distinct subsets: (1) the thrombocytopenia, anasarca, fever/elevated C-reactive protein (CRP), reticulin myelofibrosis, renal dysfunction, and organomegaly (iMCD-TAFRO) clinical subtype and (2) the iMCD–not otherwise specified (iMCD-NOS) subtype characterized by thrombocytosis, milder clinical features, and hypergammaglobulinemia. Our lab has dedicated extensive effort over the last five years to uncover several potential mechanisms for iMCD. Though nearly all of the biological work we have performed has been on circulating immune cell populations and serum, the pathological features in iMCD occur in the lymph node. Systematic unbiased investigation of lymph node tissue utilizing a targeted gene expression approach to generate hypotheses about the underlying mechanisms is urgently needed. The main goal of this project is to generate and analyze transcriptomic data to improve our understanding of key immune cell types, activated signaling pathways, and driver cytokines in iMCD.

Main Objectives:

Bulk profiling of gene expression in the lymph node of CD subtypes and related inflammatory disorders

  1. Filter high impact gene candidates for improved patient subtyping and ideal treatment options. 

    1. cross compare identified DEG’s from bulk RNAseq, serum proteomics data, scRNAseq data, and previously published lymph node data from other groups (CHOP/Mayo) to identify critical genes in iMCD subtypes. 

  2. Validation/characterization of top candidate genes in-vitro. 

    1. validate using an orthogonal platform/immunofluorescence (IF)/ and spatial gene expression methods to characterize cell types that are responsible for the dysregulated features in iMCD lymph nodes.

Research Questions:

  1. Can we recreate external analyses that were performed (and published) on lymph node tissue in MCD?

  2. Is the Wistar RNAseq data good quality? Do we see phenotype separation in PCA plot in Wistar RNAseq data? How much does changing how many variable genes are accounted for change the PCA plot?

  3. What genes and/or pathways are upregulated in iMCD-NOS and iMCD-TAFRO when compared to sentinel?

  4. How do these genes and/or pathways compare to our SPEED or other research studies?

  5. Are there genes upregulated in TAFRO vs NOS?

  6. What are the genes and/or pathways that are upregulated in iMCD (TAFRO + NOS) vs sentinel?

  7. What are the genes and/or pathways that are upregulated in SLN and SLE?

  8. How do they overlap in that we found in iMCD?

  9. What cell types are responsible for the upregulated gene that are enriched in iMCD?

  10. Are results from orthogonal gene expression platforms (nanostring vs. bulk RNAseq) comparable?